Abstract

Industrial production of polyhydroxyalkanoates (PHAs) has been in development during the last decades due to the biodegradability, biocompatibility and sustainability of these biopolymers. In addition, PHAs have shown to be a promising source for obtaining chiral chemicals from renewable carbon sources as they are composed of R-3-hydroxyalkanoic acids (R-3HAs). R-3HAs are valuable molecules used as starting materials in the chemical synthesis of antibiotics, vitamins, flavors, and pheromones. These chiral synthons are attractive compounds for many potential applications but are rarely commercially available. PHA depolymerization, either in-vivo or by hydrolytic degradation, has been proven to be a good method to obtain R-3HAs. However, as the most common mcl-PHAs are random copolymers consisting of two or more 3-HAs, the recovery and purification process of these compounds is quite tedious, expensive and requires the use of organic solvents. In this work, in a first step, an optimized bacterial fermentation of Pseudomonas putida KTQQ20 led to the polymer production (25-50 % wt PHA accumulation) and characterization (NMR and GC) of six different mcl-PHAs homopolymers. In a second step, one of the mcl-homopolymer (PHA-C10) was chosen to be evalutated by its in vivo depolymerization and the related 3-HAs release. PHA-C10 homopolymer was obtained after 15 h of batch fermentation in modified E2 medium with decanoic acid (4 mM) as substrate. Finally, cells containing 31 % wt of polymer, were harvested, washed and re-suspended in phosphate buffer (50 mM) at pH 11. Depolymerization reaction and 3HAs release was followed by HPLC and HPLC-MS. Results indicated that after 22 h 350 µg/mL of 3-hydroxydecanoate was released and 85% of the initial polymer content depolymerized. Parallel experiments indicated that when the initial polymer content in the cell was higher, the concentration of 3HA released reached 800-1400 µg/mL but the depolymerization reaction is inhibited, being only 20-25 % of the polymer depolymerized. The NMR spectra confirmed the chemical structure for the 3-OH-decanoate. First results indicate that recovery yields of up to 70-80% should be feasible. In principle, this protocol could be applied to all the mcl-homopolymers and therefore six different types of 3HAs could be produced. Currently, the methodology is being evaluated in order to obtain different 3HAs and assess their recovery yields. To conclude, we are convinced that our method of producing 3-HA from mcl-PHA homopolymers by in-vivo depolymerization has a clear benefit in simplicity over the chemical synthesis or the hitherto established in-vivo degradation of random copolymers in Pseudomonas putida GPo1, where a separation of the different 3-HAs is required.